EP1602375B1 - Administration des produits de la voies de synthèse de la 5-lipoxygenase pour la traitement d'infections microbiennes - Google Patents

Administration des produits de la voies de synthèse de la 5-lipoxygenase pour la traitement d'infections microbiennes Download PDF

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EP1602375B1
EP1602375B1 EP05016311A EP05016311A EP1602375B1 EP 1602375 B1 EP1602375 B1 EP 1602375B1 EP 05016311 A EP05016311 A EP 05016311A EP 05016311 A EP05016311 A EP 05016311A EP 1602375 B1 EP1602375 B1 EP 1602375B1
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leukotriene
composition
ltb
leukotrienes
administration
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EP1602375A3 (fr
EP1602375A2 (fr
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Marc Peters-Golden
Theodore Standiford
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University of Michigan
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

  • the present invention relates generally to a therapeutic composition
  • a therapeutic composition comprising an effective amount of a product of the 5-lipoxygenase pathway for use as a medicament for the prophylaxis or treatment of a microbial infection in patients having certain predisposing factors for a microbial infection.
  • the macrophages can elaborate chemotactic factors which recruit circulating neutrophils to the airspaces and activate their phagocytic and microbicidal activities.
  • phagocytic cells are capable by themselves of microbial ingestion, the efficiency of this process is enhanced by the presence of various soluble molecules (opsonins) which coat the organisms and mediate their attachment to surface receptors on the phagocyte.
  • opsonins include immunoglobin as well as factors which coat microbes nonspecifically, such as the complement fragment C3b, surfactant protein A, and fibronectin.
  • phagocytosis and intracellular killing are further augmented by a variety of activating agents, including colony stimulating factors, chemokines, and lipids.
  • activating agents including colony stimulating factors, chemokines, and lipids.
  • qualitative or quantitative impairment of any component of these defenses can compromise bacterial clearance and predispose to pneumonia. [ See, e.g., J Langermans et al.. J. Immunol. Methods 174:185-194 (1994) ].
  • pneumonia In industrialized countries, pneumonia is associated with greater morbidity and mortality than any other type of infection. Overall, it is the sixth leading cause of death in the United States. In adults greater than 65 years of age, it is the fourth most common reason for hospitalization. Among hospital-acquired infections, pneumonia is the second most common in incidence and the most commonly fatal.
  • Bacteria are the etiologic pathogens in a substantial proportion of community-acquired pneumonias and in the great majority of nosocomial pneumonias.
  • enteric Gram-negative organisms are the etiologic microbes responsible for both types of pneumonia.
  • Gram-negative pneumonias are generally thought to result from microaspiration of oral secretions, and are therefore particularly likely in individuals demonstrating oropharyngeal colonization with these organisms. This is especially common in hospitalized patients, particularly those in intensive care units, but also occurs in alcoholics, patients with underlying systemic illness or impairments in host defense, and those with chronic pulmonary disease. [ S. Nelson et al., Clin. Chest Med. 16:1-12 (1995) ].
  • Acquired drug resistance is usually caused by a mutation within the genome of the microbe or by the acquisition of a plasmid.
  • one of the major mechanisms of resistance to the ⁇ -lactam antibiotics, including penicillins is the production of ⁇ -lactamases.
  • resistance to one member of a class of agents e.g ., the aminopenicillin ampicillin
  • can result in complete cross-resistance to other members of that class e.g. , the aminopenicillin amoxicillin).
  • Antibiotic pressure in certain patient populations has contributed to the development of infections with multi-drug resistant organisms, the eradication of which is increasingly difficult.
  • One factor contributing to antibiotic pressure is the widespread use of antibiotics in the hospital setting, especially in the critical care units. Indeed, physicians are frequently forced to utilize antibiotic regimens comprising multiple agents to combat such infections or to use broad-spectrum agents (e.g., Primaxin®, Merck) generally reserved for the most serious infections.
  • the enhancement means should be efficacious in the treatment and prevention of bacterial pneumonia in those patients who are especially susceptible thereto, should have a rapid onset of action, and should not elicit immunological reactions in the recipient.
  • Leukotrienes are potent mediators of inflammation derived from the 5-lipoxygenase pathway of arachidonic acid metabolism. These substances have been implicated in the pathogenesis of inflammatory lung diseases, and new pharmacologic agents that inhibit leukotriene synthesis or actions have recently become available for the treatment of asthma.
  • the present invention contemplates the use of leukotriene B 4 and derivatives as in table I as an adjunct in the treatment of pneumonia and other lower respiratory tract infections in patients having a predisposing factor.
  • the present inventors have employed a model of Klebsiella pneumonia in knockout mice rendered leukotriene-deficient by the targeted disruption of the 5-lipoxygenase gene.
  • the present inventors found that leukotriene production was increased in the lungs of infected wild type mice, and that leukotriene-deficient animals manifested reduced bacterial clearance and enhanced lethality.
  • alveolar macrophages from knockout mice exhibited impaired in vitro phagocytosis and killing of K. pneumoniae, and this functional defect in leukotriene-deficient alveolar macrophages was overcome by the addition of exogenous leukotrienes such as LTB 4 .
  • intrapulmonary administration of LTB 4 partially overcame the in vivo impairment in bacterial clearance observed in knockout mice.
  • the present inventors have determined that endogenous leukotrienes play an integral role in the host response to pulmonary infection. Even more importantly from a therapeutic standpoint the present inventors found that exogenous leukotrienes exert pharmacologic actions which augment this response.
  • the present invention contemplates the treatment of patients with a recognized predisposing factor (e.g ., smoking, alcoholism, diabetes, I-IIV infection, known aspiration) for overwhelming pneumonia, or with early pneumonia, with administration via inhalation or an endotracheal tube of metabolic products of the 5-lipoxygenase pathway, wherein said microbial infection and said predisposing factor are not both an HIV infection, and wherein said product of the 5-lipoxygenase pathway comprises a leukotriene selected from the group consisting of leukotriene B4, leukotriene B4-d4, leukotriene B4 dimethyl amide, 6-trans leukotriene B4, 6-trans-12-epi leukotriene B4, 12-epi leukotriene B4, 18-carboxy dinor leukotriene B4, 20-carboxy leukotriene B4, and 20-hydroxy leukotriene B4.
  • a recognized predisposing factor e.g ., smoking
  • the present invention contemplates the use of the products of the 5-lipoxygenase pathway for prophylactic purposes. While an understanding of the mechanism by which the products act is not necessary for the successful practice of the present invention, the administration of such products, especially the intrapulmonary administration of leukotrienes, augments local endogenous host defense mechanisms and assists in bacterial infection eradication during antibiotic administration.
  • the products have a relatively short duration of action ( e.g ., hours), do not cause antibody-mediated immune responses, and are relatively inexpensive.
  • the present invention is not limited to the intra-pulmonary administration of products of the 5-lipoxygenase pathway for the treatment of pneumonia. Indeed, the present invention contemplates the administration of these products via other routes of administration and for the treatment and prevention of other conditions.
  • the products may be administered concomitantly with antibiotics in some embodiments.
  • different products e.g ., LTB 4
  • different products e.g ., LTB 4
  • the present invention contemplates a therapeutic composition comprising an effective amount of a product of the 5-lipoxygenase pathway for use as a medicament for the treatment of a microbial infection in a patient having a predisposing factor for a microbial infection, said predisposing factor being smoking, alcoholism, diabetes, HIV infection, AIDS virus, vitamin D deficiency or being in the neonatal period; with the proviso that said microbial infection and said predisposing factor are not both an HIV infection, wherein said product of the 5-lipoxygenase pathway comprises a leukotriene selected from the group consisting of leukotriene B4, leukotriene B4-d4, leukotriene B4 dimethyl amide, 6-trans leukotriene B4, 6-trans-12-epi leukotriene B4, 12-epi leukotriene B4, 18-carboxy dinor leukotriene B4, 20-carboxy leukotriene B4, and 20-hydroxy leu
  • the present invention contemplates a therapeutic composition
  • a therapeutic composition comprising an effective amount of a product of the 5-lipoxygenase pathway for use as a medicament for the prophylaxis of a microbial infection in a patient having a predisposing factor for a microbial infection, said predisposing factor being smoking, alcoholism, diabetes, HIV infection, AIDS virus, vitamin D deficiency or being in the neonatal period; with the proviso that said microbial infection and said predisposing factor are not both an HIV infection, wherein said product of the 5-lipoxygenase pathway comprises a leukotriene selected from the group consisting of leukotriene B4, leukotriene B4-d4, leukotriene B4 dimethyl amide, 6-trans leukotriene B4, 6-trans-12-epi leukotriene B4, 12-epi leukotriene B4, 18-carboxy dinor leukotriene B4, 20-carboxy leukot
  • the microbial infection is bacterial pneumonia.
  • the method of administering comprises pulmonary administration, and the pulmonary administration is by aerosolization of the therapeutic composition in other embodiments.
  • certain embodiments further involve the co-administration of an antibiotic to the host.
  • the host is an animal in some embodiments, and a human in others.
  • the present invention contemplates a therapeutic composition for use in treating a bacterial infection, comprising administering an effective amount of the therapeutic composition to a host having a bacterial infection, the therapeutic composition comprising a leukotriene.
  • the bacterial infection is bacterial pneumonia.
  • the leukotriene is leukotriene B 4 in certain embodiments.
  • the method of administering comprises pulmonary administration, and the pulmonary administration is by aerosolization of the therapeutic composition in other embodiments.
  • certain embodiments further involve the co-administration of an antibiotic to the host.
  • the host is an animal in some embodiments, and a human in others.
  • the present invention contemplates a solution for the treatment of a microbial infection, the solution comprising a sterile liquid vehicle and a leukotriene dissolved in the sterile liquid vehicle, wherein said solution is aerosolized, and wherein said leukotriene is selected from the group consisting of leukotriene B 4 , leukotriene B 4 -d 4 , leukotriene B 4 dimethyl amide, 6-trans leukotriene B 4 , 6-trans-12-epi leukotriene B 4 , 12-epi leukotriene B 4 , 18-carboxy dinor leukotriene B 4 , 20-carboxy leukotriene B 4 , and 20-hydroxy leukotriene B 4 .
  • the leukotriene is leukotriene B 4 .
  • product of the 5-lipoxygenase pathway refers to those compounds that result from the enzymatic conversion of arachidonic acid by 5-lipoxygenase.
  • Products of the 5-lipoxygenase pathway include 5-hydroperoxyeicosatetraenoic acid [5-HPETE] and LTA 4 , as well as compounds derived therefrom.
  • the products encompass 5-HETE, which is produced from 5-HPETE.
  • the products also include compounds formed from the conversion of LTA 4 , such as LTB 4 , LTC 4 , LTE 4 , and LTF 4 .
  • the products are meant to encompass derivatives ( i . e ., compounds produced by structural modification) of compounds produced in the arachidonic acid cascade.
  • leukotriene is herein defined functionally as those compounds causing enhancement of antimicrobial defense.
  • microbial includes, but is not limited to, bacteria, viruses, parasites, and fungi.
  • cysteine residue characteristic of leukotrienes C 4 , D 4 , and E 4 refers to those leukotrienes that possess the cysteine residue characteristic of leukotrienes C 4 , D 4 , and E 4 .
  • eicosanoid refers to compounds derived from 20-carbon essential fatty acids that contain three, four, or five double bonds: 8,11,14-eicosatrienoic acid (dihomo- ⁇ -linolenic acid), 5,8,11,14-eicosatetraenoic acid (arachidonic acid), and 5,8,11,14,17-eicosapentaenoic acid.
  • the families of leukotrienes and prostaglandins are examples of eicosanoids.
  • the term "effective amount" refers to that amount of a 5-lipoxygenase product that is required to successfully perform a particular function.
  • the effective amount of a 5-lipoxygenase product will be that amount that enhances or improves (to any degree) the ability of the body to eradicate a microbial infection, especially a bacterial infection.
  • the effective amount may depend on a number of factors, including the type of microbe involved, the severity of the infection, the immune status of the individual, and the weight of the individual.
  • leukotriene LTD 4 may be administered in a therapeutic composition containing between 0.1 ⁇ g and 10 ⁇ g.
  • the term "therapeutic composition” refers to a composition that comprises a product of the 5-lipoxygenase pathway (e.g., LTB 4 and LTC 4 ) in a pharmaceutically acceptable form.
  • the characteristics of the form will depend on a number of factors, including the mode of administration.
  • a composition for aerosolized pulmonary administration must be formulated such that the product is pharmacologically active following delivery to the lungs.
  • the therapeutic composition may contain diluents, adjuvants and excipients, among other things.
  • the product of the 5-lipoxygenase pathway is dissolved in a sterile liquid vehicle.
  • sterile liquid vehicle refers to those liquids that are suitable for administration to a host ( e.g. , pulmonary or parenteral administration) and allow dissolution of the product of the 5-lipoxygenase pathway. Examples of sterile liquid vehicles include, but are not limited to, sterile normal saline and dilute concentrations of ethanol.
  • the term "host” refers to humans and animals.
  • enhancing microbial defense and “enhancing bacterial defense” refer broadly to the improved ability of a subject's immune system to respond to and eradicate a microbial infection (e.g. , a bacterial, parasitic, viral, and fungal infection) and specifically a bacterial infection, respectively.
  • a microbial infection e.g. , a bacterial, parasitic, viral, and fungal infection
  • the terms include, for example, augmentation of the subject's endogenous defense mechanisms.
  • the presence of enhancement of antimicrobial/antibacterial defense is determined by subjecting a compound to the screening procedure described in Table 3 below.
  • the present invention relates generally to the administration of products of the 5-lipoxygenase pathway, wherein said product of the 5-lipoxygenase pathway comprises a leukotriene selected from the group consisting of leukotriene B4, leukotriene B4-d4, leukotriene B4 dimethyl amide, 6-trans leukotriene B4, 6-trans-12-epi leukotriene B4, 12-epi leukotriene B4, 18-carboxy dinor leukotriene B4, 20-carboxy leukotriene B4, and 20-hydroxy leukotriene B4, to enhance microbial defense, and more particularly to the administration of the products of the 5-lipoxygenase metabolic pathway to enhance bacterial defense and to treat and prevent bacterial pneumonia.
  • leukotriene B4-d4 leukotriene B4 dimethyl amide
  • 6-trans leukotriene B4 6-trans-12-epi leukotriene B4, 12-epi leukotriene B4, 18-car
  • Leukotrienes are oxygenated derivatives of arachidonic acid synthesized mainly by bone marrow-derived cells in response to a variety of soluble or particulate stimuli. [ E. Goetzl et al., FASEB J 9:1051-1058 (1995) ].
  • Arachidonic acid is initially hydrolyzed from membrane phospholipids, in part by the actions of cytosolic phospholipase A 2 (cPLA 2 ).
  • the next two steps in leukotriene synthesis (the sequential conversion of arachidonic acid first to 5-hydroperoxyeicosatetraenoic acid [5-HPETE] and then to LTA 4 ) are catalyzed by the enzyme 5-lipoxygenase (5-LO).
  • 5-LO translocates in a Ca 2+ -dependent manner to the nuclear envelope [ see, e.g., J. Woods et al., J. Clin. Invest. 95:2035-2040 (1995) ]; here it is thought to gain access to free arachidonic acid, hydrolyzed from nuclear envelope phospholipids and presented by the integral nuclear envelope arachidonic acid-binding protein, 5-LO activating protein (FLAP).
  • 5-HPETE can be converted to the stable product, 5-HETE.
  • the LTA 4 can be enzymatically converted to LTB 4 (by LTA 4 hydrolase) or to LTC 4 (by LTC 4 synthase).
  • LTC 4 can be enzymatically converted to LTD 4 (with concomitant increase in bioactivity) and then to LTE 4 ; LTE 4 may be subsequently modified to form LTF 4 .
  • FIG. 1A is a schematic depicting the pathway of leukotriene synthesis and the structures of the main products of the 5-lipoxygenase metabolic pathway; importantly, practice of the present invention does not depend on the accuracy of the model depicted in FIG. 1A .
  • Cellular leukotriene synthetic capacity can be enhanced by exposure to a number of biologically active substances, such as granulocyte-macrophage colony-stimulating factor, interferon- ⁇ , and transforming growth factor- ⁇ .
  • biologically active substances such as granulocyte-macrophage colony-stimulating factor, interferon- ⁇ , and transforming growth factor- ⁇ .
  • alveolar macrophages have a greater capacity for 5-LO metabolism than do blood monocytes or other tissue macrophages [ see, e.g., M. Peters-Golden et al., J. Immunol. 144:263-270 (1990) ], and they produce both LTB 4 and LTC 4 .
  • Neutrophils by contrast, produce only LTB 4 .
  • Alveolar macrophages and neutrophils both produce 5-HETE.
  • LTB 4 is a potent neutrophil chemotaxin in vitro, accounting for the majority of chemotactic activity elaborated acutely by stimulated human alveolar macrophages in culture. [ T. Martin et al., J. Clin. Invest. 80:1114-1124 (1989) ]. In addition, in vivo bronchoscopic instillation of LTB 4 into the human lung resulted in neutrophil influx. [ T. Martin et al., J. Clin. Invest. 80:1009-1019 (1989) ].
  • LTB 4 is thought to enhance numerous leukocyte functions, including phagocytosis [ T. Demitsu et al., Int. J. Immunopharmac. 11:801-808 (1989) ], upregulation of cell surface CR3 molecules [ P. Marder et al., Biochem. Pharmacol. 49:1683-1690 (1995) ], the secretion of O 2 - and lysosomal hydrolases, mobilization of intracellular Ca 2+ stores [ C. Serhan et al., Biochem. Biophys. Res. Commun.
  • the phagocyte surface receptors which are most critical for efficient opsonic phagocytosis are those which recognize the Fc portion of IgG (FcRII and FcRIII) and the C3bi fragment of complement (the integrin CR3, also known as Mac-1 and CD11b/CD18).
  • CR3 also mediates nonopsonic ingestion of K. pneumoniae .
  • One consequence of receptor ligation is the release and metabolism of arachidonic acid. Because CR3 and FcR mediate attachment of K. pneumoniae to phagocytes, their surface expression are relevant targets for modulation by leukotrienes.
  • FIG. 1B is a schematic depicting the pathway of leukotriene synthesis and the actions of leukotrienes relevant to antimicrobial defense.
  • Bacteria such as K . pneumoniae attach to phagocytic cells such as alveolar macrophages and neutrophils and are phagocytosed. It is believed that this triggers an increase in intracellular Ca 2+ , which in turn results in translocation of cPLA 2 and 5-LO to the nuclear envelope.
  • arachidonic acid is hydrolyzed from phospholipids and metabolized by 5-LO, interacting with FLAP, to LTA 4 .
  • LTA 4 is further converted to leukotrienes B 4 and C 4 .
  • These may affect target cells, via interactions with receptors, in either autocrine or paracrine fashion. As a result, chemotaxis, bacterial phagocytosis, and bacterial killing are promoted.
  • exogenously administered products of the 5-LO pathway in general, and leukotrienes in particular are associated with a number of possible advantages as adjunctive agents in the treatment of pneumonia.
  • the inventors have determined that these products exhibit a rapid onset of action and believe that these products do not elicit immunologic responses in the recipient.
  • such products represent a relatively inexpensive therapy that can be used independent of antibiotics or as adjunct therapy to antibiotics in the treatment of pneumonia.
  • Particular patient populations e.g ., patients with AIDS, diabetes, smokers, neonates, and patients suffering from alcoholism and malnutrition
  • severe pneumonia would benefit from augmenting endogenous host defense mechanisms through the rational administration of, for example, leukotrienes to the lungs.
  • Klebsiella pneumoniae is the classic cause of Gram-negative pneumonia and has been reported to account for 18-64% of community-acquired and 30% of nosocomial Gram-negative pneumonias. [ L. Crane and A. Lemer, In: Respiratory Infections: Diagnosis and Management (J. Pennington. ed.) (Raven Press, New York), pp. 227-250 (1983 )].
  • LTB 4 enhances microbial phagocytosis and/or killing.
  • the addition of LTB 4 promotes neutrophil chemotaxis as well as phagocytosis of particles, signal transduction, and secretion of oxidants and lysosomal enzymes - all of which would be expected to facilitate bacterial clearance.
  • LTB 4 enhanced the in vitro phagocytosis and killing of P. aeruginosa and Salmonella typhimurium by peritoneal macrophages [ T. Demitsu et al., Int. J. Immunopharmac.
  • Alveolar macrophages have been demonstrated to have a greater capacity for leukotriene synthesis than peripheral blood monocytes or other tissue macrophages. This is the situation in response to both soluble (ionophore A23187) and particulate (zymosan) agonists, and for cells from humans [ M. Balter et al., J. Immunol. 142:602-608 (1989) ] as well as rats [ M. Peters-Golden et al., J. Immunol. 144:263-270 (1990) ] (data not shown). Moreover, as described further in the Experimental section, the profile of eicosanoids released by stimulated murine alveolar macrophages is likewise comprised largely of 5-LO metabolites ( see FIG. 2A ).
  • the present inventors have also demonstrated that neutrophils recruited to sites of inflammation exhibit increased leukotriene synthetic capacity and a shift in intracellular 5-LO distribution. Indeed, the present inventors have compared leukotriene synthetic capacity and intracellular distribution of 5-LO in rat neutrophils isolated from peripheral blood or from peritoneal lavage fluid 4 hours after glycogen instillation. Elicited neutrophils exhibited a 5-fold greater maximal capacity for LTB 4 synthesis in response to A23187 than did blood neutrophils studied in parallel (data not shown). In addition, the two cell populations exhibited strikingly different intracellular distributions of 5-LO in the resting state. As previously demonstrated for human blood neutrophils [ T.G. Brock et al., J. Biol. Chem.
  • the resting rat blood neutrophils contained 5-LO exclusively in the cytosol.
  • the resting elicited neutrophils contained a substantial proportion of their 5-LO within the nucleus; upon subsequent ionophore activation, both blood and elicited neutrophil populations showed 5-LO translocation to the nuclear envelope (data now shown).
  • the present inventors used Klebsiella pneumoniae as a causative pathogen to induce pneumonia for several reasons. First, as previously discussed, it is of great clinical relevance in pneumonia. Second, it causes a brisk inflammatory response in mice. [ A. McColm et al., J. Antimicrob. Chemother. 18:599-608 (1986) ]. Third, the murine K. pneumoniae model has been extensively characterized by one of the co-inventors. In the experiments described below, intratracheal (i.t.) injection was utilized rather than aerosolization because it more closely resembles the bolus of organisms which reaches the distal lung via microaspiration. Following intratracheal challenge of CD-1 mice with 10 3 CFU of K.
  • neutrophil influx peaks at 48 hours and most animals have died by day 5.
  • lung homogenate levels of various cytokines increase and also peak at 48 hours; these include tumor necrosis factor (TNF), macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein-1 ⁇ (MIP-1 ⁇ ), IL-12, and IL-10.
  • TNF tumor necrosis factor
  • MIP-2 macrophage inflammatory protein-2
  • MIP-1 ⁇ macrophage inflammatory protein-1 ⁇
  • IL-12 IL-12
  • IL-10 IL-10
  • the present invention utilizes a murine K. pneumonia model
  • the present invention is not limited to augmenting the treatment of infections caused by that organism.
  • the present invention contemplates the administration of products of the 5-LO metabolic pathway, wherein said product of the 5-lipoxygenase pathway comprises a leukotriene selected from the group consisting of leukotriene B4, leukotriene B4-d4, leukotriene B4 dimethyl amide, 6-trans leukotriene B4, 6-trans-12-epi leukotriene B4, 12-epi leukotriene B4, 18-carboxy dinor leukotriene B4, 20-carboxy leukotriene B4, and 20-hydroxy leukotriene B4, particularly LTB 4 , independently and as an adjunct ( e.g ., with antibiotics) to the treatment of pneumonia and other respiratory tract infections caused by a panoply of organisms.
  • an adjunct e.g ., with antibiotics
  • Table 2 lists some of the most common bacterial pathogens that cause community-acquired and hospital-acquired pneumonia. It is contemplated that patients with infections caused by these organisms will benefit from administration of the products of the 5-LO metabolic pathway.
  • Type of Pneumonia Type of Pathogen Community most frequent: Streptococcus pneumoniae Haemophilus influenzae Mycoplasma pneumoniae less frequent: Staphylcoccus aureus Legionella sp.
  • the present invention is not limited to augmentation of the treatment of pneumonia. Indeed, the present invention contemplates the administration of products of the 5-LO metabolic pathway as therapy in the treatment of other infections that have pulmonary manifestations. Moreover, as alluded to above, the present invention contemplates the administration of the products for the treatment and prophylaxis of a broad range of microbial infections besides bacterial infections, including inflections caused by parasites [ R. Moqbel et al., Clin. Exp. Immunol. 52:519-527 (1983) ], viruses, and fungi. Furthermore, the present invention contemplates augmentation of the treatment of systemic infections; it should be pointed out that systemic administration should be performed cautiously, as the leukotrienes are known to cause hypotension.
  • anti-leukotriene drugs are likely to mimic the leukotriene deficiency observed with 5-LO gene disruption in mice.
  • the use of such drugs may compromise pulmonary antimicrobial host defense.
  • these individuals may also benefit from administration of products of the 5-LO pathway contemplated for use with the present invention; of course, particular dosing schedules and regimens may be warranted when these agents are used concomitantly with patients taking anti-leukotriene drugs.
  • the present invention contemplates the use of diverse products of the 5-lipoxygenase metabolic pathway in order to enhance bacterial defense.
  • the comprehensive screening procedure set forth in Table 3 can be used to evaluate those products (such as those compounds previously presented in Table 1), as well as derivatives or analogues of such products, that may be effective.
  • Leukotrienes B 4 and C 4 are particularly effective at enhancing bacterial defense, and this screen is especially appropriate for compounds related to those leukotrienes.
  • Reference to a particular example is given with each determination; the indicated examples provide a detailed description of how the determination is to be carried out.
  • Step II Measure in vitro the activity of compounds on alveolar macrophage phagocytic and bactericidal activities ( see , e . g ., Example 3). Proceed to Step II with those compounds that increase phagocytic and bactericidal activities. II Determine in vivo the effect of compounds on bacterial clearance after 48 hours by measuring CFU in lung homogenate ( see , e . g ., Example 2). Proceed To Step III with those compounds that increase clearance. III Determine in vivo the effect of compounds via different routes of administration and administered at different time points post-bacterial challenge ( see , e.g., Example 10). Proceed to Step IV with those compounds that exhibit efficacy following administration via at least one route. IV Verify the findings of Step III by examining animal survival. Consider clinical trials.
  • compositions of products of the 5-LO metabolic pathway that are indicated as being efficacious based on application of the screen described above. It is not intended that the present invention be limited by the particular nature of the therapeutic preparation.
  • such compositions can be provided together with physiologically tolerable liquid (e.g., saline), gel or carriers or vehicles, diluents, adjuvants and excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like, and combinations thereof.
  • physiologically tolerable liquid e.g., saline
  • diluents e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like, and combinations thereof.
  • These compositions typically contain 1%-95% of active ingredient, preferably 2%-70%.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or
  • therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents.
  • dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts and the organism involved.
  • a preferred mode of administration comprises administration to the lung.
  • Patients who are sick enough to require mechanical ventilation can receive treatment with pharmacologic agents administered via the endotracheal tube which is connected to the ventilator.
  • intrapulmonary delivery of pharmacologic agents to patients not requiring mechanical ventilation can be accomplished via aerosolization.
  • the agent may be administered to the lung through a bronchoscope.
  • the therapeutic agents may be investigated for their efficacy via other routes of administration, including parenteral administration. However, when the site of infection is the lung, targeting drug delivery thereto is likely to minimize side effects and systemic consequences.
  • the compounds contemplated by the present invention possess attributes as therapeutic agents over other agents like polypeptides.
  • the products of the 5-LO metabolic pathway contemplated by the present invention have a rapid onset of action (generally within 1 hour) and short duration of action (generally less than 12 hours); these attributes permit a substantial degree of control over biological effects.
  • their short duration of action reduces the possibility that administration of leukotrienes and related agents might adversely stimulate an over-exuberant inflammatory response.
  • commercially-available leukotriene receptor antagonists e.g., the cysteinyl antagonist Accolate® (zafirlukast) Zeneca
  • the products of the 5-LO metabolic pathway contemplated by the present invention are associated with additional attributes.
  • the lipid products do not elicit immunologic reactions like polypeptide agents do.
  • the compounds of the present invention are relatively inexpensive, making them ideal as an adjunct to infection treatment.
  • the compounds contemplated by the present invention provide a means for enhancing pulmonary defense capabilities. They are especially efficacious in the treatment and prevention of bacterial pneumonia in those patients who are predisposed to that condition.
  • the present invention contemplates the use of the compounds in the treatment and prevention of other infections and ailments, alone or in combination with, for example, other products of the 5-LO pathway or antimicrobial agents.
  • M M
  • mM millimolar
  • ⁇ M micromolar
  • N Normal
  • mol molecular weight
  • mM millimolar
  • ⁇ M micromolar
  • pmol picomoles
  • g grams: mg (milligrams); ⁇ g (micrograms); L (liters); mL (milliliters); ⁇ L (microliters); cm (centimeters); mm (millimeters); ⁇ m (micrometers); nm (nanometers); min. (minutes); s and sec.
  • IL Biogenics
  • Cayman Cayman Chemical; Ann Arbor, MI); Coulter (Coulter Corp., Miami, FL); Difco (Detroit, MI); Fisher Scientific. Pittsburg, PA); Gibco (Gibco BRL; Gaithersburg, MD); Jackson (The Jackson Laboratory; Bar Harbor, ME); Merck (Rahway, NJ); Molecular Probes (Eugene, OR); Nunc (Naperville, IL); PharMingen (San Diego, CA); Pierce (Rockford, IL); Pfizer (Pfizer Inc., New York, NY); Vector (Vector Laboratories, Burlingame, CA); and Waters (Waters Corp., Milford, MA); Zeneca (Zeneca Pharmaceuticals, Wilmington, DE).
  • mice with the targeted disruption of their 5-LO gene ALOX 5, designated KO
  • their wild type strain controls 129/SvEv, designated WT
  • K. pneumoniae strain 43816, serotype 2 obtained from the ATCC (Assession No. 29939) was grown in tryptic soy broth (Difco) for 18 hours at 37°C.
  • the preparation and intratracheal administration of K. pneumoniae were carried out as described by M. Schneemann et al. [J. Infect. Dis. 167:1358-1363 (1993) ].
  • Bacterial concentration was determined by measuring absorbance at 600 nm and referencing to a standard curve of absorbances vs. known standard CFUs. Bacteria were then pelleted by centrifugation for 30 min at 10.000 rpm, washed x 2 in saline, and resuspended at the desired concentration in saline.
  • mice After appropriate dilution of bacteria in endotoxin-free saline, animals were anesthetized with sodium pentobarbital (approximately 0.2 mL diluted 1:7 in saline intraperitoneally) and the trachea was exposed via a small midline incision. A 30 ⁇ L inoculum containing 50 CFU K. pneumoniae or saline was administered via a sterile 26-gauge needle and the skin was closed with a surgical staple.
  • K. pneumoniae -specific serum wild type mice are similarly anesthetized and inoculated intratracheally (with 25 CFU bacteria); animals are bled orbitally 2 weeks later, and serum obtained.
  • Plasma and lung CFU were determined as described by M. Schneemann et al. [J. Infect. Dis. 167:1358-1363 (1993) ]. Briefly, lungs homogenized in 3 mL sterile saline and plasma collected at euthanasia were placed on ice, and serial 1:10 dilutions made. Ten ⁇ L of each dilution were plated on soy base blood agar plates (Difco), incubated for 18 hours at 37°C, and colonies were enumerated.
  • mice were anesthetized and blood was collected by orbital exsanguination. The mice were then euthanized via cervical dislocation and whole lungs were harvested for the determination of cytokine levels, myeloperoxidase activity (MPO), and leukotriene levels.
  • MPO myeloperoxidase activity
  • lungs were homogenized in 2 mL of buffer containing 0.5% Triton X-100, 150 mM NaCl, 15 mM Tris-HCl, 1 mM CaCl 2 , and 1 mM MgCl 2 .
  • the homogenate was sonicated and centrifuged and the supernatant was mixed 1:15 with assay buffer (86 mM monobasic sodium phosphate, 12 mM dibasic sodium phosphate, 0.0005% [v/v] H 2 O 2 , and 0.167 mg/mL o -dianisidine hydrochloride) and read at 490 nm (Beckman DU-64). MPO units were calculated as the change in absorbance over time. Protein content of homogenates is determined using a microtiter plate modification (Pierce Biochemical) of the Bradford method using bovine serum albumin as a standard.
  • the trachea was exposed through a 0.5 cm incision and intubated using a 1.7 mm OD polyethylene catheter. Bronchoalveolar lavage was performed by instilling 1 mL aliquots of phosphate-buffered saline containing 5 mM EDTA. Approximately 4 mL of lavage fluid were retrieved per mouse, and total cell numbers and differential cell counts were determined from cytospins on each sample.
  • alveolar macrophages were purified from bronchoalveolar lavage cells by adherence for 1 hour in HBSS and studied in monolayer culture. Adherent cells were preincubated with 5% K pneumoniae -specific immune serum (as a source of both complement and specific opsonizing antibody) for 5 minutes at 37°C prior to assays. Phagocytosis was studied by incubating 10 5 alveolar macrophages with 10 6 K . pneumoniae in each well of an 8-well Labtek® plate (Nunc) for 1 hour at 37°C; in some experiments, exogenous LTB 4 (Cayman) was added concomitantly with bacteria.
  • K pneumoniae -specific immune serum as a source of both complement and specific opsonizing antibody
  • the supernatants were aspirated and the cells were washed 3 times with HBSS.
  • the slides were then allowed to air dry, Diff-Quik® (Difco) staining was performed, and 200 cells per well were counted to determine number of intracellular K. pneumoniae and percent of alveolar macrophages containing bacteria. Phagocytic index was calculated as the mean percentage of alveolar macrophages containing bacteria multiplied by the mean number of bacteria per alveolar macrophage.
  • the bactericidal activity was assayed by incubating for 1 hour at 37°C the same numbers of alveolar macrophages and organisms as detailed above, but in 35 mm tissue culture dishes. Supernatants were removed and cells were than washed with HBSS and lysed by adding 1 mL of cold sterile water, scraping with a rubber policeman, and incubating on ice for 10 minutes. One mL of 2x HBSS was added to each plate and lysates were serially diluted on blood agar plates. Plates were incubated for 18 hours at 37°C and colony counts performed.
  • Percent killing of intracellular bacteria was calculated by the following formula: 100 - (number of bacterial CFU/mL alveolar macrophage lysate divided by the total number of intracellular bacteria), where total intracellular K. pneumoniae is the product of the total number of alveolar macrophages x the percentage of alveolar macrophages containing bacteria x the mean number of bacteria per alveolar macrophage.
  • mice are injected intraperitoneally with 5% glycogen in PBS and peritoneal lavage is performed 5 hours later. Approximately 3 x 10 6 cells are obtained from each animal, approximately 85-90% of which are neutrophils. These cells are likewise placed into culture for functional studies. Phagocytic and bactericidal assays are performed as described above.
  • Lung lavage from K. pneumoniae -challenged animals yields a mixture of alveolar macrophages and neutrophils; they are found in a ratio of approximately 1:1 at 2 days post-inoculation, but the ratio is likely to vary over time.
  • mixed bronchoalveolar lavage cells are placed into culture (5 x 10 5 cells/well) as described above for purified populations; the ratio of alveolar macrophage:neutrophil in adherent monolayers is determined by direct Diff-Quik® staining of monolayers after the removal of medium. In all instances where leukotriene levels in culture medium are quantitated, values are expressed per ⁇ g of cell protein.
  • Culture medium is medium 199 (Gibco).
  • Ethanolic stock solutions of LTB 4 , LTC 4 , and 5-HETE were diluted in saline and a 10 ⁇ L volume used for intratracheal injection.
  • a particle size ⁇ 3 ⁇ m and a nose-only exposure chamber is utilized.
  • Sections and cytospins are incubated at 4°C for 24 hours with either rabbit anti-human 5-LO antiserum (Merck Frosst Canada) or nonimmune rabbit serum at 1:1000 in 25% normal goat serum in PBS. This antibody also recognizes the mouse and murine 5-LO. Goat anti-rabbit IgG (1:600) is then applied for 30 minutes and primary antibody is detected using True-Blue® peroxidase substrate with Contrast Red® counterstain (both from KPL Laboratories). The proportion of positively stained cells exhibiting an activated pattern is determined from counts of 20 high power fields. Cells staining positively for 5-LO (most of which are expected to be either macrophages or neutrophils) are classified as to cell type on the basis of morphology.
  • Cell type-specific staining is accomplished either with a second primary (e.g., anti-neutrophil antibody) or via histochemical staining (e.g, for nonspecific esterase or MPO).
  • the second protein is detected by Vector Red® (Vector) to contrast with the True-Blue® stain for 5-LO.
  • CR3 and FcR are quantitated in both alveolar macrophages and neutrophils by staining with FITC-conjugated anti-mouse monoclonal antibodies with subsequent analysis by flow cytometry.
  • the FITC-conjugated monoclonal antibodies include anti-CR3 IgG 1 , anti-FcRII/FcRIII IgG 1 , and an anti-IgG, isotype control. Experimental incubations are carried out in suspension.
  • Engulfment of attached particles or bacteria requires cytoskeletal rearrangement, including local actin polymerization.
  • Polymerized actin (F-actin) is analyzed by staining with rhodamine-phalloidin (Molecular Probes) at a 1:300 dilution. Intracellular localization of F-actin is assessed by immunofluorescence microscopy. Cells on cover slips are fixed with formalin and permeabilized in acetone. [ T.G. Brock et al., J. Biol. Chem. 269:22059-22066 (1994) ]. Following incubation with phalloidin for 1 hour, the cells are examined with a Nikon Labophot 2 microscope equipped for epifluorescence.
  • Adherent cells from knockout or wild type mice are prelabeled by incubation for 15 minutes with 5 ⁇ g/mL acridine orange (Molecular Probes). Cells are washed, preincubated with specific immune serum, and then incubated for up to 2 hours with K . pneumoniae alone or in the presence of exogenous leukotrienes. Cells are examined by immunofluorescence microscopy. Two hundred cells per condition are counted, and the percentage of cells showing fusion as well as the total number of fusion figures are recorded.
  • acridine orange Molecular Probes
  • FIGS. 2A and B depict RP-HPLC profiles of radioactive eicosanoids released by prelabeled alveolar macrophages obtained from wild type mice ( FIG. 2A ) and 5-LO knockout mice ( FIG. 2B ).
  • the profiles were obtained by prelabeling 10 6 alveolar macrophages overnight with [ 3 H]arachidonic acid. The alveolar macrophages were then washed and stimulated for 30 minutes with 1 ⁇ M A23187. The medium was subjected to lipid extraction and radiolabeled eicosanoids separated by reverse-phase HPLC. Peaks were identified on the basis of co-elution with authentic standards. As compared to cells from wild type control animals ( FIG.
  • alveolar macrophages from KO animals produced no leukotrienes or 5-HETE, as expected.
  • FIG. 3 graphically depicts the effect of K. pneumoniae challenge on survival in 5-LO knockout mice (solid circles) and wild type mice (open squares) (*p ⁇ 0.05 vs. WT).
  • administration of 50 CFU of bacteria led to 60% mortality in wild type mice within 8 days, with no subsequent deaths thereafter.
  • all of the knockout mice died in response to this same challenge, with all deaths occurring by day 10.
  • deaths in the knockout group occurred earlier than in the wild type animals.
  • mean lung as well as plasma CFUs were almost two logs greater in knockout mice than in wild type mice at 48 hours post-challenge.
  • the proportion of knockout animals that developed bacteremia at this time point (15/19) was significantly greater than that of wild type mice (10/19).
  • 66% of knockout mice were bacteremic (average plasma CFU of 1.06 x 10 5 ), while no wild type mice had bacteria in their plasma at this time point (data not shown).
  • the experiments of this example assess the ability of the alveolar macrophages themselves, the first line of cellular defense, to phagocytose and kill K. pneumoniae in vitro and the effect of administering exogenous leukotrienes on alveolar macrophage antibacterial functions in vitro.
  • Alveolar macrophages were purified by adherence of bronchoalveolar lavage cells lavaged from uninfected knockout and wild type animals, and preincubated for 5 minutes with 5% K. pneumoniae-specific immune serum (as a source of both complement and specific opsonizing antibody) prior to assays. Cultured alveolar macrophages from either group of mice were incubated in the presence of specific serum with K. pneumoniae for 1 hour and then washed, after which monolayers were either stained with Diff-Quik (Difco) and intracellular organisms enumerated, or lysed and bacterial CFUs in lysates determined following overnight culture. Phagocytic index and intracellular killing were calculated as detailed above in General Methods.
  • FIG. 5 graphically depicts phagocytic and bactericidal activities in alveolar macrophages isolated from 5-LO knockout mice (cross-hatched bars) and wild type mice (solid bars); in FIG. 5 , each value represents the mean ⁇ SEM of 6 replicate cultures (*p ⁇ 0.05 vs. WT). As indicated by the data in FIG. 5 , alveolar macrophages from 5-LO knockout mice demonstrated significant decreases in their abilities to both ingest and kill K pneumoniae when compared to cells from wild type mice.
  • FIG. 6 graphically depicts the effect of exogenous LTB 4 (none, 0.1 nM, and 5 nM LTB 4 added) on bacterial phagocytic activity in alveolar macrophages from 5-LO KO mice; each value represents the mean from triplicate cultures.
  • LTB 4 dose-dependently enhanced the phagocytic index in knockout alveolar macrophages, with an index approximately three times the baseline level at a concentration of 5 nM.
  • neutrophils manifest similar functional defects in phagocytosis and killing which could contribute to the sensitivity to bacterial pneumonia seen in knockout mice in vivo .
  • exogenous LTB 4 The effects of exogenous LTB 4 on phagocytosis by neutrophils from 5-LO knockout mice were also examined. Glycogen-elicited neutrophils were obtained from the peritoneal cavity of knockout mice, and phagocytosis of K. pneumoniae over a one hour time period was evaluated in the presence and absence of exogenous LTB 4 (1 nM); under these circumstances, phagocytic index was 27 ⁇ 8 and 45 ⁇ 4, respectively (data not shown). These results with exogenous LTB 4 are important in several respects. First, they indicate that the phagocytic defect in these cells is actually related to the deficiency of 5-LO, and is not coincidental.
  • both IL-12 and TNF have been shown to play critical protective roles in this model of murine pneumonia. Additionally, TNF production is potentiated by leukotrienes in some experimental systems. To examine the possibility that the enhanced susceptibility of knockout mice to bacterial challenge might relate to an impaired ability to generate either of these cytokines, lung homogenates were analyzed 48 hours after bacterial challenge. Again, no significant differences were found in antigenic IL-12 or TNF levels between infected knockout and wild type mice (data not shown). Thus, the increased lethality of pneumonia in 5-LO knockout mice does not reflect diminished capacity to produce these pro-inflammatory cytokines.
  • exogenous LTB 4 increased the phagocytic index of 5-LO knockout alveolar macrophages by approximately 300%, more than would have been necessary to merely attain the control level manifested by wild type cells (approximately 50% increase). That result indicates that the leukotriene is exhibiting a pharmacological effect.
  • the experiments of this example further evaluate the effects of exogenous LTB 4 on phagocytic capacity of normal alveolar macrophages and examine the effects of other 5-LO products besides LTB 4 .
  • FIG. 9 graphically depicts the effect of the exogenous 5-LO metabolites on bacterial phagocytic activity in normal rat alveolar macrophages.
  • Each value in FIG. 9 represents the mean ⁇ SEM of 4 replicate cultures.
  • LTB 4 evoked an approximately 6-fold increase in phagocytic index in normal rat alveolar macrophages.
  • the metabolite 5-HETE had a similar, though less pronounced, effect.
  • LTC 4 augmented phagocytosis to a degree similar to LTB 4 .
  • cysteinyl leukotrienes like LTC 4 have been observed to upregulate surface FcR expression in macrophages, increased phagocytic capacity has not been noted previously.
  • LTB 4 was administered together with the intratracheal inoculum of K. pneumoniae (50 CFU). A dose of 6 ng of LTB 4 intratracheally per animal was chosen for two reasons. First, other researchers previously found that this dose and route resulted in a brisk neutrophil influx 6 hours after administration in mice. [ N. Ahmed et al., Am J. Respir. Crit. Care Med. 153:1141-1147 (1996) ]. Second, the present inventors previously found ( see FIG 5 ) that approximately 7 ng of total LTB 4 could be measured in the homogenate of a pair of lungs from Klebsiella -challenged wild type mice.
  • FIG. 10 graphically depicts the effect of intratracheal administration of LTB 4 on defective bacterial clearance of the lung in 5-LO knockout mice (each value represents the mean ⁇ SEM).
  • the data shown in FIG. 10 confirm the previous finding ( FIG. 4 ) that knockout mice had approximately 100-fold more organisms in their lungs than did wild type animals: it should be noted that the absolute CFUs in this experiment were less because analysis was performed at 24 hours after inoculation rather than 48 hours.
  • the single intratracheal dose of LTB 4 administered concomitantly with the bacterial inoculum reduced the lung CFU by approximately 10-fold in knockout mice.
  • the results indicate that exogenous LTB 4 is capable of augmenting pulmonary clearance of K. pneumoniae in these leukotriene-deficient mice.
  • leukotrienes should be effective therapeutic agents in the setting of Gram-negative pneumonia.
  • the examples described above employing intratracheal Klebsiella challenge in 5-LO knockout mice demonstrate that the enzyme plays an in vivo role in pulmonary antibacterial host defense.
  • the experiments of this example are directed at ascertaining the roles and mechanisms of action of 5-LO products in the host response to K. pneumoniae using knockout mice as well as mice treated with pharmacological agents which inhibit leukotriene synthesis or actions. More specifically, the experiments of this example are directed at discerning the role of LTB 4 vs.
  • cysteinyl leukotrienes by comparing the effects of a variety of pharmacologic agents, including those which target both classes of leukotrienes (5-LO inhibitor), those which target only LTB 4 (LTB 4 receptor antagonist), and those which target only cysteinyl leukotrienes (cysteinyl leukotriene receptor antagonists).
  • pharmacologic agents including those which target both classes of leukotrienes (5-LO inhibitor), those which target only LTB 4 (LTB 4 receptor antagonist), and those which target only cysteinyl leukotrienes (cysteinyl leukotriene receptor antagonists).
  • mice with 50 CFU of K pneumoniae are utilized in the experiments of this example.
  • wild type mice are treated with various long-acting agents (set forth below) by the oral (gavage) route, with daily dosing commencing the morning of the day before the administration of bacteria.
  • oral (gavage) route with daily dosing commencing the morning of the day before the administration of bacteria.
  • the specificity of the agents to be used has been established, and the selection of doses and dosing regimens is guided by published experience in rodents.
  • a single maximally effective dose of each drug is determined from assessments made at 24 hours after initiation of treatment.
  • the specific agents and preliminary dose ranges which are tested include the following: i) the 5-LO inhibitor A-79175 (Abbott) in a 1-3 mg/kg dose; this is a competitive enzyme inhibitor that is a more potent and longer-acting congener of Zileuton® with demonstrated efficacy in mice as a once-daily oral agent; ii) the LTB 4 antagonist CP-105,696 (Pfizer) in a 1-10 mg/kg dose; this compound has inhibited collagen-induced arthritis in mice when administered in a once-daily oral dose; and iii) the LTD 4 antagonist MK-571 (Merck) in a 0.1-1 mg/kg dose; this compound has effectively inhibited antigen-induced bronchoconstriction when administered orally to rats.
  • the 5-LO inhibitor A-79175 Abbott
  • this is a competitive enzyme inhibitor that is a more potent and longer-acting congener of Zileuton® with demonstrated efficacy in mice as a once-d
  • LTB 4 antagonism is assessed by quantitating the ex vivo LTB 4 -stimulated upregulation of CR3 expression on neutrophils in whole blood obtained from drug-treated animals. Cysteinyl leukotriene receptor antagonism is assessed by quantitating Evans blue dye extravasation following intradermal administration of LTD 4 . [ J. Drazen et al., Proc. Natl. Acad. Sci USA 77:4354-4358 (1980) ].
  • bacterial CFU is determined in whole lung homogenates and plasma obtained from animals sacrificed at both 1 day and 3 days post- Klebsiella challenge.
  • lung neutrophil influx is initially assessed by MPO activity of whole lung homogenates from the same animals used for CFU determinations above; if MPO assays suggest that active drug treatment results in a reduction in neutrophil influx, an additional experiment is carried out (since lavage and homogenization cannot be performed in the same animal) in which such an effect is verified by bronchoalveolar lavage cell counts and differentials on drug- vs. vehicle-treated animals.
  • determining the relative contribution to host defense of endogenously synthesized LTB 4 versus LTC 4 allows i) the design of therapeutic studies employing administration of exogenous leukotrienes and ii) the assessment of possible risks to infection susceptibility of, for example, 5-LO inhibitors (which inhibit synthesis of LTB 4 and cysteinyl leukotrienes in parallel) and cysteinyl leukotriene receptor antagonists (which selectively inhibit the actions of cysteinyl leukotrienes without affecting those of LTB 4 ).
  • time points are selected for further studies designed to determine the cellular sources of leukotrienes through i) immunohistochemical staining in order to identify cells exhibiting an intracellular distribution of 5-LO associated with enzyme activation, and ii) measuring constitutive leukotriene production by cells isolated from pneumonic lungs.
  • mice are inoculated intratracheally with either saline or with 50 CFU of K. pneumoniae , and lungs are harvested at 8 hours and 1, 2, 3, 5, and 7 days post-inoculation.
  • lung sections are prepared for immunohistochemistry (see below).
  • bronchoalveolar lavage cytospins are prepared and levels of leukotrienes are determined in cell-free lavage fluid.
  • Levels of LTB 4 and LTC 4 in lavage fluid and in lung homogenates are correlated with each other and with the degree of neutrophil influx (assessed from MPO activity in homogenates and cell counts and differentials from bronchoalveolar lavage fluid cytospins).
  • the cellular sources of leukotriene production in the lung is determined on the 8 hour, 1 day, and 3 day time points and other time points identified by the above kinetic analysis indicating maximal levels of leukotrienes B 4 or C 4 .
  • Immunohistochemical staining for 5-LO is performed on lung sections along with bronchoalveolar lavage cytospin preparations from both Klebsiella- and saline-challenged mice in order to determine whether it is the alveolar macrophages, neutrophils, or both cell types which demonstrate an intracellular distribution of 5-LO characteristic of enzyme activation (i.e., staining concentrated at the nuclear envelope).
  • Determining 5-LO activation in lung tissue in situ by this method has the advantage that it does not require cell isolation or culture, obviating concerns about the potential artifacts which might be introduced by those procedures.
  • this approach has been used in idiopathic pulmonary fibrosis to demonstrate that alveolar macrophages isolated by bronchoalveolar lavage from patients with idiopathic pulmonary fibrosis constitutively overproduce leukotrienes when placed into culture, even in the absence of an exogenous agonist. [ J. Wilborn et al., J. Clin. Invest. 97:1827-1836 (1996) ].
  • studying mixed cell populations should not create difficulty in attributing leukotriene generation to a particular cell type at the 8 hour time point because there is a relatively pure population of alveolar macrophages at the time.
  • alveolar macrophages and neutrophils synthesize unique profiles of leukotriene products; thus, alveolar macrophages produce primarily LTC 4 ( Fig. 2A ) while neutrophils synthesize primarily LTB 4 .
  • the profile of leukotrienes elaborated by cultured bronchoalveolar lavage cells provides strong evidence for the involvement of each cell type.
  • studying mixed lavage cells allows potential neutrophil-alveolar macrophage interactions in leukotriene synthesis to take place, as they inevitably do in vivo.
  • LTB 4 vs. LTC 4 Knowledge of the kinetics of endogenous production of LTB 4 vs. LTC 4 is helpful in several important respects. First, it provides guidance in designing the "therapeutic" experiments (described below) involving pulmonary administration of exogenous leukotrienes. Second, determining the contributions of alveolar macrophages and neutrophils as sources for the production of these mediators provides basic information about the biology of the host response. Finally, knowledge of the appropriate cellular sources of leukotrienes in the setting of bacterial pneumonia has potential diagnostic utility in that documenting deficient leukotriene production may help to identify patients who may be candidates for exogenous pulmonary leukotriene supplementation in order to augment innate immunity.
  • the experiments of this example elucidate the molecular mechanisms by which specific 5-LO metabolites enhance phagocytosis and killing. More specifically, the experiments of this example involve adding different lipids to alveolar macrophages and elicited neutrophils obtained both from knockout mice and from wild type mice in order to compare the magnitude of effects and mechanisms of action for different 5-LO products in both cell types. These experiments provide a means of i) further evaluating the therapeutic utility of leukotrienes, and ii) evaluating the utility of particular molecular and/or biochemical markers as endpoints to be examined in the in vivo leukotriene treatment studies described below in Example 10.
  • Elicited neutrophils are studied instead of peripheral blood neutrophils because of the possibility that the process of recruitment and/or residence in an inflammatory milieu itself alters cellular phenotype.
  • cells obtained from both wild type and knockout mice are studied.
  • the mechanistic endpoints for study are as follows: i) surface expression of receptors necessary for binding/ingestion of K. pneumoniae (assessed by flow cytometry), including FcRII/FcRIII and CR3; ii) actin microfilament assembly (assessed by immunofluorescent staining and flow cytometry), necessary for particle engulfment; and iii) phagosome-lysosome fusion (assessed by acridine orange staining), necessary to bring the microbe in contact with the bactericidal arsenal.
  • the General Methods describes the procedures for each of these assessments.
  • Bactericidal mechanisms are examined in a manner similar to that described for phagocytosis. Again, while an understanding of the molecular mechanisms is not required in order to practice the present invention, subsequent experiments are performed to address the molecular mechanism(s) that are responsible for 5-LO metabolites that augment killing of K. pneumoniae . Moreover, the ability of antagonists to block the positive effects of lipids on these mechanistic events are evaluated, and alveolar macrophages and elicited neutrophils are both studied. Three bactericidal mechanisms are examined (as described in the General Methods section). First, extracellular generation of O 2 - is assessed by the superoxide dismutase-inhibitable reduction of ferricytochrome C. [ L.
  • the LTB 4 antagonist CP-105,696 and the cysteinyl leukotriene antagonist MK-571 are used. They are added to wild type cells prior to addition of K. pneumoniae, and phagocytosis, killing, and relevant molecular mechanisms are then evaluated as described above.
  • the data obtained from the preceding examples provides, among other things, information regarding the time point in the host response at which the presence of particular leukotrienes is most critical.
  • the experiments of this example use that information to rationally test the in vivo efficacy of exogenous leukotrienes, either singly or in combination, administered by different routes.
  • the initial experiments of this example involve animals whose endogenous capacity for leukotriene generation is impaired because of 5-LO gene disruption. Subsequent experiments test the efficacy of intrapulmonary leukotriene administration in wild type mice. Finally, in addition to the clinically relevant endpoints of bacterial clearance and survival, the experiments of this example investigate the utility of profiling a molecular consequence of leukotriene action (e.g. , CR3 expression) on lavaged cells as a possible surrogate for predicting diminished (without exogenous leukotrienes) or enhanced (with exogenous leukotrienes) bacterial clearance and survival.
  • a molecular consequence of leukotriene action e.g. , CR3 expression
  • 5-LO knockout mice are used for the first series of studies. Knockout mice are given 50 CFU of K. pneumoniae intratracheally together with LTB 4 in doses ranging from 1-20 ng per animal (6 ng was the dose utilized in the experiment corresponding to FIG. 10 ); a similar dose range of LTC 4 is also tested. Lung and plasma bacterial CFUs are determined at 1 day, and the results of these experiments are used to determine optimal doses of concomitantly administered LTB 4 and LTC 4 .
  • leukotriene(s) administered together with the bacterial inoculum should augment the bacterial clearance potential of the alveolar macrophage.
  • administering leukotriene(s) at a later time point is associated with other potential merits. For example, activation of the recruited neutrophils might be accomplished if active compound is dispensed at approximately 1-3 days post-inoculation.
  • an efficacious post-inoculation regimen is more readily applicable to treatment of overwhelming Gram-negative pneumonia in patients.
  • the first regimen entails early ( e . g ., with inoculation) and late ( e.g., day 2) administration.
  • the early and late 5-LO metabolite can be selected independent of each other; in other words, LTB 4 can be utilized for one dose and LTC 4 for the other dose.
  • the second regimen entails continuous administration of leukotriene(s) by aerosol.
  • leukotrienes are nebulized and administered to mice via a nose-only exposure chamber. Selection of the metabolite and the treatment window (e.g. , days 1-3) is based on the results from the one-time dosing experiments.
  • leukotriene-deficient mice are applied to K. pneumoniae -challenged wild type mice.
  • 129/SvEv wild type mice are more susceptible to Klebsiella pneumonia than are many other strains, although not as susceptible as 5-LO knockout mice.
  • These wild type mice may therefore be more closely representative of patients susceptible to Gram-negative pneumonia than are the leukotriene-deficient animals. Therefore, the optimal leukotriene treatment strategy defined from studies in knockout mice is used in wild type mice, with similar endpoints of bacterial clearance and survival.
  • the experiments disclosed in this example indicate the effects of aerosolized and intratracheal administration of post- Klebsiella challenge on bacterial clearance and survival in both wild type and 5-LO knockout mice. These experiments serve to provide information regarding the in vivo administration of exogenous leukotrienes.
  • the studies described involve treatment with leukotrienes B 4 and C 4 ; these were selected because of their known actions and their potency.
  • the use of other 5-LO products, including 5-HETE and lipoxins is contemplated by the present invention.

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Claims (21)

  1. Composition thérapeutique comprenant une quantité efficace d'un produit de la voie de la 5-lipoxygénase destinée à être utilisée comme médicament pour la prophylaxie ou le traitement d'une infection microbienne chez un patient présentant un facteur prédisposant à une infection microbienne, ledit facteur prédisposant étant le tabac, l'alcoolisme, le diabète, l'infection par le VIH, le virus du sida, la déficience en vitamine D ou la période néonatale ;
    à condition que ladite infection microbienne et ledit facteur prédisposant ne soient pas tous deux l'infection par le VIH, où ledit produit de la voie de la 5-lipoxygénase comprend un leucotriène choisi dans le groupe consistant en leucotriène B4, leucotriène B4-d4, diméthylamide du leucotriène B4, 6-trans leucotriène B4, 6-trans-12-épi leucotriène B4, 12-épi leucotriène B4, 18-carboxy dinor leucotriène B4, 20-carboxy leucotriène B4 et 20-hydroxy leucotriène B4.
  2. La composition de la revendication 1, dans laquelle ledit patient est caractérisé par le fait qu'il présente un facteur prédisposant choisi dans le groupe consistant en tabac, alcoolisme et diabète.
  3. La composition de la revendication 1, dans laquelle une infection à VIH a été diagnostiquée chez ledit patient présentant un facteur prédisposant.
  4. La composition de la revendication 1, dans laquelle ledit produit de la voie de la 5-lipoxygénase comprend un leucotriène choisi dans le groupe consistant en leucotriène B4 et 20-hydroxy leucotriène B4.
  5. La composition de la revendication 4, dans laquelle ledit leucotriène est le leucotriène B4.
  6. La composition de la revendication 1, dans laquelle ladite administration comprend une administration par voie pulmonaire.
  7. La composition de la revendication 6, dans laquelle ladite administration par voie pulmonaire se fait par aérosolisation de ladite composition thérapeutique.
  8. La composition de la revendication 1, dans laquelle ladite prophylaxie ou ledit traitement comprend également la co-administration d'un antibiotique au dit patient.
  9. La composition de la revendication 1, dans laquelle ledit patient est un animal.
  10. La composition de la revendication 1, dans laquelle ledit patient est un humain.
  11. La composition de la revendication 1, dans laquelle ladite infection microbienne est une infection bactérienne.
  12. La composition de la revendication 11, dans laquelle ledit patient est caractérisé par le fait qu'il présente un facteur prédisposant choisi dans le groupe consistant en tabac, alcoolisme et diabète.
  13. La composition de la revendication 11, dans laquelle une infection à VIH a été diagnostiquée chez ledit patient.
  14. La composition de la revendication 11, dans laquelle ledit produit de la voie de la 5-lipoxygénase comprend un leucotriène choisi dans le groupe consistant en leucotriène B4 et 20-hydroxy leucotriène B4.
  15. La composition de la revendication 14, dans laquelle ledit leucotriène est le leucotriène B4.
  16. La composition de la revendication 11, dans laquelle ladite administration comprend une administration par voie pulmonaire.
  17. La composition de la revendication 16, dans laquelle ladite administration par voie pulmonaire se fait par aérosolisation de ladite composition thérapeutique.
  18. La composition de la revendication 11, comprenant également la co-administration d'un antibiotique au dit patient.
  19. La composition de la revendication 11, dans laquelle ledit patient est un animal.
  20. La composition de la revendication 11, dans laquelle ledit patient est un humain.
  21. Solution destinée à être utilisée dans le traitement d'une infection microbienne, ladite solution comprenant un véhicule liquide stérile et un produit de la voie de la 5-lipoxygénase dissous dans ledit véhicule liquide stérile, où ladite solution est aérosolisée, et où ledit produit de la voie de la 5-lipoxygénase comprend un leucotriène choisi dans le groupe consistant en leucotriène B4, leucotriène B4-d4, diméthylamide du leucotriène B4, 6-trans leucotriène B4, 6-trans-12-épi leucotriène B4, 12-épi leucotriène B4, 18-carboxy dinor leucotriène B4, 20-carboxy leucotriène B4 et 20-hydroxy leucotriène B4.
EP05016311A 1996-12-03 1997-12-03 Administration des produits de la voies de synthèse de la 5-lipoxygenase pour la traitement d'infections microbiennes Expired - Lifetime EP1602375B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/757,136 US5909734A (en) 1996-12-03 1996-12-03 Administration of products of the 5-lipoxygenase metabolic pathway to enhance antimicrobial defense
US757136 1996-12-03
EP97954070A EP1037640B1 (fr) 1996-12-03 1997-12-03 Administration de produits par la voie metabolique de la 5-lipogenase pour renforcer les defenses antimicrobiennes

Related Parent Applications (1)

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EP97954070.5 Division 1998-06-11

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EP1602375A2 EP1602375A2 (fr) 2005-12-07
EP1602375A3 EP1602375A3 (fr) 2005-12-14
EP1602375B1 true EP1602375B1 (fr) 2010-02-17

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EP05016311A Expired - Lifetime EP1602375B1 (fr) 1996-12-03 1997-12-03 Administration des produits de la voies de synthèse de la 5-lipoxygenase pour la traitement d'infections microbiennes
EP97954070A Expired - Lifetime EP1037640B1 (fr) 1996-12-03 1997-12-03 Administration de produits par la voie metabolique de la 5-lipogenase pour renforcer les defenses antimicrobiennes

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EP97954070A Expired - Lifetime EP1037640B1 (fr) 1996-12-03 1997-12-03 Administration de produits par la voie metabolique de la 5-lipogenase pour renforcer les defenses antimicrobiennes

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US (3) US5909734A (fr)
EP (2) EP1602375B1 (fr)
AT (2) ATE457730T1 (fr)
AU (1) AU733567B2 (fr)
CA (1) CA2278140C (fr)
DE (2) DE69739775D1 (fr)
DK (1) DK1037640T3 (fr)
ES (2) ES2341255T3 (fr)
WO (1) WO1998024397A2 (fr)

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US7473708B2 (en) 1996-02-15 2009-01-06 Jean Gosselin Agents with leukotriene B4-like antiviral (enveloped RNA) activities
US5909734A (en) * 1996-12-03 1999-06-08 Regents Of The University Of Michigan Administration of products of the 5-lipoxygenase metabolic pathway to enhance antimicrobial defense
KR100325581B1 (ko) * 1998-08-07 2002-08-24 오우택 아라키토닉산의리폭시게네이즈대사결과물질을함유하는진통제용조성물
WO2004112795A1 (fr) * 2003-06-25 2004-12-29 Ltb4 Sweden Ab Compositions de ltb4 pour le traitement d'infections du tractus respiratoire
US20050239889A1 (en) * 2004-04-26 2005-10-27 Jean Gosselin In vivo release of endogenous anti-microbial mediators by leukotriene B4 (LTB4) administration
EP1796730B1 (fr) * 2004-09-20 2010-07-21 LTB4 Sweden AB Preparation pharmaceutique contenant le leucotriene b4 (ltb4) stabilise
EP3791880A1 (fr) 2009-04-29 2021-03-17 Amarin Pharmaceuticals Ireland Limited Compositions pharmaceutiques comprenant de l'epa
CN112546196A (zh) * 2020-02-29 2021-03-26 中国人民解放军总医院第三医学中心 肌动蛋白聚合诱导剂在制备抵抗或抑制肺炎克雷伯氏菌的药物中的应用

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Also Published As

Publication number Publication date
AU5794298A (en) 1998-06-29
EP1037640A4 (fr) 2003-07-23
ATE457730T1 (de) 2010-03-15
AU733567B2 (en) 2001-05-17
ES2341255T3 (es) 2010-06-17
WO1998024397A3 (fr) 1998-09-03
CA2278140A1 (fr) 1998-06-11
EP1602375A3 (fr) 2005-12-14
ES2251748T3 (es) 2006-05-01
EP1037640B1 (fr) 2005-10-26
EP1602375A2 (fr) 2005-12-07
US7696148B1 (en) 2010-04-13
EP1037640A2 (fr) 2000-09-27
DK1037640T3 (da) 2006-03-13
DE69739775D1 (de) 2010-04-01
US5909734A (en) 1999-06-08
DE69734476T2 (de) 2006-07-13
DE69734476D1 (de) 2005-12-01
CA2278140C (fr) 2008-02-05
US20050267211A1 (en) 2005-12-01
WO1998024397A2 (fr) 1998-06-11
ATE307589T1 (de) 2005-11-15

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